Do Haplogroups H and U Act to Increase the Penetrance of Alzheimer’s Disease?

Abstract

1. Alzheimer’s disease (AD) is the most common form of dementia in the elderly in which interplay between genes and the environment is supposed to be involved. Mitochondrial DNA (mtDNA) has the only noncoding regions at the displacement loop (D-loop) region that contains two hypervariable segments (HVS-I and HVS-II) with high polymorphism. mtDNA has already been fully sequenced and many subsequent publications have shown polymorphic sites, haplogroups, and haplotypes. Haplogroups could have important implications to understand the association between mutability of the mitochondrial genome and the disease.

2. To assess the relationship between mtDNA haplogroup and AD, we sequenced the mtDNA HVS-I in 30 AD patients and 100 control subjects. We could find that haplogroups H and U are significantly more abundant in AD patients (P = 0.016 for haplogroup H and P = 0.0003 for haplogroup U), Thus, these two haplogroups might act synergistically to increase the penetrance of AD disease.

INTRODUCTION

Alzheimer’s disease [(AD)(MIM 104300)] is a progressive, degenerative disease of the brain that causes a person to forget recent events and familiar tasks. AD is the most common form of dementia in the elderly. Approximately 5% of AD cases are familial (FAD) with autosomal-dominant transmission; resulting mainly from mutations in the Amyloid β Precursor Protein (ABPP) or in the ABPP processing proteins presenilin-1(PS1) and (PS2). Most patients with AD are sporadic cases (SAD) without a known genetic defect. There are several disparate lines of evidence pointing to mitochondrial involvement in the disease (Smith et al., 2002; Coskun et al., 2004).

Current research has shown that there are two distinct differences in the brains of AD patients when compared with non-AD patients. First, formation of senile plaques that disrupt the normal structure and function of nerve cells. Second, formation of neurofibrillary protein tangles that cause the branches that sprout from neurons to become disorganized and collapse (Bennett et al., 2004). This lack of structure results in severed neuronal connections that cause the dementia (Bennett et al., 2004). The cause of sporadic AD remains enigmatic (Harman, 2002). One hypothesis for the etiology of late-onset, sporadic AD is that it is caused by defects in mitochondrial oxidative phosphorylation (OXPHOS) (Coskun et al., 2004). Structurally abnormal mitochondria have been observed in AD brains, and deficiencies in mitochondrial OXPHOS enzymes, such as cytochrome c oxidase (COX or Complex IV) have been repeatedly reported in the brains and other tissues of AD patients (Bosetti et al., 2002; Cottrell et al., 2002). Defects in OXPHOS inhibit ATP production, but they also increase mitochondrial reactive oxygen species (ROS) production, which, in turn, can activate the mitochondrial permeability transition pore (mtPTP) and destroy the surrounding cell by apoptosis (Coskun et al., 2004).

The human mitochondrial genome (mtDNA) is a small 16569 kb molecule of double-stranded DNA. It has noncoding regions at displacement loop region (D-loop) that contain three hypervariable segments (HVS-I, HVS-II, HVS-III) with high polymorphism. Most of the mutations observed in both mtDNA coding and noncoding regions have occurred on preexisting haplogroups and define the individual mtDNA types or haplotypes (Torroni et al., 1996; Graven et al., 1995). Haplogroups could have important implications for understanding of the relationship between mutability of the mitochondrial genome and disease (Ozawa et al., 1991; Shoffner et al., 1993; Lertrit et al., 1994; Obermaier-Kusser et al., 1994). There is growing evidence that certain mtDNA clusters are associated with distinct disorders (Brown et al., 1995; Torroni et al., 1997; Hassani-Kumleh et al., 2006). To examine the involvement of mtDNA haplogroups in determining susceptibility to AD, we sequenced the mtDNA HVS-I in 30 patients with AD and 100 normal subjects.

PATIENTS AND METHODS

Patients

To study the relationship between AD patients and mtDNA haplogroups, we sequenced HVS-I from 30 Iranian patients (13 females and 17 males) suffering Alzheimer’s disease aged 52–76 years and 100 normal controls aged 56–75 years. The male-to-female ratio was 1.3:1 for patients and 1.2:1 for normal controls.

All of the patients and controls were informed on the aims of the study and gave their informed consent to the genetic analysis. Average ages of the patients and controls were 63±12 and 68±9, respectively. All of the patients had probable Alzheimer’s disease according to the National Institute of Neurological and Communicative Diseases and Stroke criteria distributed by the Long Island Alzheimer’s foundation. The controls were randomly chosen from people who had no AD symptoms or family history of it. Control people had been evaluated by a neurologist to exclude any findings of dementia and other neurological disorders.

Patients had a complete physical exam, along with a detailed history of symptoms and medical history, including medication. Clinical examinations were done by a neurologist. At the time of blood sampling among 30 patients, 17 (7 females and 10 males) were on moderate stage of the disease. Familial forms of Alzheimer’s disease were excluded. Patients who had hypertension, diabetes mellitus, and history of stroke were excluded as well. All of the patients had brain atrophy in magnetic resonance imaging (MRI) without any mass lesion or marked vascular imaging signs such as stroke or intracerebral hemorrhage. The patients had been checked for infections or conditions such as B12 deficiency, anemia, thyroid function abnormalities, renal, hepatic, and other causes of memory loss. Psychiatric assessment to uncover possible depression or other mental illness were done. All of the patients had an electroencephalography for recognizing special abnormalities.

mtDNA Haplogroup

Peripheral blood samples were obtained and DNA was purified after lyses of white blood cells by use of DNA extraction kit (Diatom DNA Extraction Kit, Genefanavaran, Tehran). PCR amplification was carried out in a final volume of 50 μL containing 200–300 ng total DNA, 10 pmol each primers, 2.5 mM MgCl₂, 200 μM of each dNTP, and 2 units Taq DNA polymerase (Roche Applied Science). Primers were as follows: Primer ONPF206 (15340-15360 nt) 5′- ATC CTT GCA CGA AAC GGG ATC -3′ and primer ONPR 77 (110-91 nt) 5′- GCT CGG GCT CCA GCG CTC CG-3′. These primers amplified a 1366 bp sequence encompassing HVS-I in the D-Loop of the mtDNA to fetch the 359 bp sequence (16024-16383 nt) for HVS I. The nucleotide sequence of the amplicon was directly determined by automated sequencing 3700 ABI machine, using primer ONPR 77 (Macrogene, Seoul, Korea). The obtained mtDNA sequences were aligned with a multiple sequence alignment interface CLUSTAL_X with comparison to rCRS (http: /www.gen.emory.edu/mitomap/ mitoseq.html).

Haplotypes were assigned to hgs according to the West Eurasian mtDNA genealogy (Macaulay et al., 1999). Hg assignment proceeded by using the following algorithm (all numbering is according to ref. (Anderson et al., 1981) minus 16,000 in the control region for brevity): 069T 126C 223C assigned to hg J; 126C 223C 294T assigned to T; 129A 223T 391A assigned to I; 223T 292T assigned to W; 189C 223T 278T assigned to X; 223C 224C 311C assigned to K; 362C assigned to D; 290T and 319A assigned to A; 223T assigned to R; 304C assigned to H1, 189C and 356T assigned to H3, 129A assigned to H4, 221T assigned to H5; 162G assigned to H8; 223C 249C and either 189C or 327T assigned to U1; 129C 223C assigned to U2: 223C 343G assigned to U3; 223C 356C assigned to U4; 223C 270T assigned to U5; 172C 219G 223C assigned to U6; 223C 318T assigned to U7; 223C 298C assigned to V; 067T 223C assigned to HV1; 126C 223C 362C assigned to preHV; 145A 176G 223T assigned to N1b; 223T 278T 390A assigned to L2; and 187T 189C 223T 278T 311C assigned to L1.

Statistical Analysis

Fisher’s exact probability test was used to examine the association between two groups. Values of P < 0.05 were regarded as statistically significant.

RESULTS

The mtDNA haplogroups of 30 AD patients and 100 normal subjects were characterized by direct sequencing of mtDNA HVS-I. Our results for the distribution of mtDNA haplogroups among 30 AD patients and 100 normal controls are summarized in Table I. Haplogroups H and U were significantly more abundant in AD patients (P = 0.016 for haplogroup H and P = 0.0003 for haplogroup U), but not for other haplogroups. Our study revealed high proportion of haplogroup H (16.6%) and U (26.6%) in patients compared to normal controls (3%).

Table I.

Distribution of mtDNA Haplogroups Among AD Patients and Normal Controls

mtDNA Haplogroups	AD Patients	Normal controls	P-Value
H	5/30 (16.6%)	3/100 (3%)	*0.016
D	5/30 (16.6%)	12/100 (12%)	0.540
L	5/30 (16.6%)	14/100 (14%)	0.769
J	4/30 (13.3%)	20/100 (20%)	0.592
T	2/30 (6.6%)	16/100 (16%)	0.242
U	8/30 (26.6%)	3/100 (3%)	*0.0003
K	1/30 (3.3%)	0/100 (0%)	0.230
R	5/30 (16.6%)	21/100 (21%)	0.795
*Others	6/30 (20%)	11/100 (11%)	0.221

^*Others: Haplogroup I, W, X, and V.

DISCUSSION

The major challenge for medical genetics appears to be the identification of genes contributing to heredity in disorders that are not transmitted in a simple Mendelian fashion. Disorders such as AD, Parkinson disease (PD), or multiple sclerosis (MS) may be polygenic. mtDNA has already been fully sequenced (Anderson et al., 1981) and many subsequent publications have revealed polymorphic sites, haplogroups, and haplotypes. The studies of mtDNA polymorphism have been based either on sequence variation in the control region, usually only the HVS-I segment, or RFLP analysis of the coding region (Weissman, 1995; Torroni et al., 1996; Macaulay et al., 1999). The sequences of HVS I can be correlated with the restriction site variants of the haplogroups H, D, L, J, T, U, K, W, and A. This correlation between nucleotide changes in mtDNA HVS I and in the coding region of mtDNA permits a direct comparison of all published data obtained by both haplotype analysis and HVS I sequencing.

Distribution of various disease groups among these haplogroups showed some of mitochondrial haplogroups to be predisposed to disease (Hofmann et al., 1997). Substitutions in the D-loop may be part of a haplotype with mutations elsewhere in the mtDNA. Mutations in mtDNA HVS-I may cause energy deficiency in stressful situations during a vulnerable developmental period (Arnestad et al., 2002). The hypothesis is that on their own, some polymorphisms are selectively neutral but that in specific combinations, they act in a synergistic, deleterious manner with established pathogenic mtDNA mutations to increase the risk of disease expression or to produce a more severe clinical outcome.

The accumulation of nonsynonymous changes in some mtDNA haplogroups can modulate either the efficiency in coupling respiration rate to ATP synthesis or the production of reactive oxygen species during respiration, or both. These effects could play a role in determining neuronal damage. We screen HVS-I to assess correlation between AD patients and normal subjects. To the best of our knowledge, this is the first study to trace mtDNA HVS-I variants in AD patients of Persian population. Our results showed that haplogroups H and U are significantly more abundant in AD patients (P = 0.016 for haplogroup H and P = 0.0003 for haplogroup U) (Table I). Thus, mtDNA haplogroups H and U might constitute a risk factor for AD. Our results for the haplogroup U is consistent with previous study in which European male samples classified as haplogroup U showed an increase in the risk of AD (van der Walt et al., 2004). We suggest that variations within haplogroup H and U may be involved in AD expression in combination with environmental exposures. Defects of mitochondrial function can result in excessive production of ROS, formation of the PTP, and release of small proteins that trigger the initiation of apoptosis, such as cytochrome c and apoptosis-inducing factor (AIF), from the mitochondrial intermembrane space into the cytoplasm. Such mitochondrial dysfunction has been proposed as a potential mechanism in the development and pathogenesis of AD, and neuronal apoptosis has been detected in AD brain. In conclusion, our data suggests association of haplogroup H and U with AD in Persian population. However, further investigations on haplotype groups and other genes may shed new light on the molecular pathogenesis of AD.